In molecular biology, PCR is a technique to amplify a single or few copies of a piece of Deoxyribonucleic Acid (DNA) by several orders of magnitude, generating millions of copies of a particular DNA sequence. This method is based on thermal cycling, i.e. continuous heating and cooling of the sample. As PCR progresses, the DNA generated is itself used as a template for replication. PCR involves three major steps of heating and cooling that is repeated for 25-30 times. Each cycle comprises of (i) denaturation at ˜94 degrees when all the double strands melt into single stranded DNA, (ii) annealing at lower temperature ˜54 degrees when both the primers pair up with single stranded templates and (iii) extension at ˜72 degrees where the polymerase extends the primer by attaching the bases complementary to the template, thus making two copies of double stranded DNA for each template.
The conventional bench-top PCR systems use large metal heating and cooling blocks that cycle the temperature of samples loaded inside polypropylene tubes, the miniaturization of the reaction chamber offers advantage in terms of integration, speed and efficiency. With these advantages the development of miniaturized systems for PCR has become an area of active interest. Devices have been fabricated by many research groups using various materials that are suitable for biological reactions. Most of the research groups have been using silicon as their substrates for its high thermal conductivity and well characterized processing conditions. Bare silicon is not optically transparent and has been reported to inhibit PCR reactions. Oxidized silicon or glass is preferable, but the complex multi-step fabrication of the reaction chambers, heaters and temperature sensors in these chips makes them too expensive for single-use disposable applications. In addition, a reaction chamber sealing step is required to avoid thermal evaporation, which is typically carried out by one of the different types of bonding such as fusion bonding, anodic bonding, adhesive bonding, reversible bonding and ultrasonic bonding. On the other hand, polymers are bio-compatible low cost materials which are transparent and can be easily molded at lower temperatures. Their thermal conductivity, however, compared to silicon, is much lower. Therefore the design of heaters and temperature sensors is important.
The choice of heating method plays an important role along with the choice of material for fast microchip PCR protocols. There have been generally two types of heating methods commonly used by various groups: contact and non contact heating. The contact heating method utilizes a resistive heater to heat up the PCR solution. The heaters are typically a thin film metal, mostly Platinum (Pt) due to its reproducible temperature dependence of resistance, ability to withstand high temperature, good chemical stability and high purity. In addition a thin layer of titanium is often used as an adhesive layer for Pt, since the latter exhibits a high diffusion rate at high temperature, which will deteriorate its performance. However Pt is very expensive and optically opaque. Other metals and alloys have also been used as heaters. Some commercially available peltier block thermo-electric units have also been widely used in temperature control of PCR chips in spite of their larger thermal mass, slower temperature ramping rates and being non transparent. Most commonly used non contact heating schemes for PCR are based on hot air cycling which is carried out by rapidly switching streams of air at the desired temperature. However the control and application of hot air for single chips may not be easy. In some reports, infrared radiation using a tungsten lamp was utilized as a non contact source of heating which needed less than 15 min for 35 cycles. However the tungsten lamp is a non-coherent and non-focused light source, and therefore needs high power (50 to 100 W) for the chip to reach the required temperature. In another report, an inexpensive halogen lamp as a low power radiation source for rapid temperature ramping in silicon micro-reaction chambers was described.
In light of forgoing discussion, it is necessary to develop non-contact real-time micro-Polymerase Chain Reaction (PCR) system which has inductively heated polymer and cite chip made of a material selected from group comprising but not limited to Poly-Dimethyl-Siloxine [PDMS], acrylic, polypropylene and polycarbonate with sealed reaction chamber and infrared temperature sensing to overcome the above mentioned problems.